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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/10—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person
  • A61K41/17—Inactivation or decontamination of a medicinal preparation prior to administration to an animal or a person by ultraviolet [UV] or infrared [IR] light, X-rays or gamma rays
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
  • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
  • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
  • A61M1/3681—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by irradiation
  • A61M1/3683—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by irradiation using photoactive agents

Definitions

• This invention is generally in the area of methods for the treatment of viral diseases, and more specifically relates to the treatment of AIDS using thiazine dyes.
• Methylene blue 3,7-Bis(dimethylamino)-phenothiazin-5-ium chloride, C 16 H 18 ClN 3 S
• Methylene blue is a dark green or blue thiazine dye which was first isolated in 1876. It is FDA approved for oral administration and has been reported to be effective as an antiseptic, disinfectant, and antidote for cyanide and nitrate poisoning.
• methylene blue is reduced by mitochondria to leukodye which is then auto-oxidized back to methylene blue by oxygen, yielding H 2 O 2 . This is the probable mechanism by which methylene blue, injected i.v.
• methemoglobinemia a clinical disorder where more than 1% of the hemoglobin in the blood has been oxidized to Fe 3+ .
• Kelner and Alexander reported in J. Biol. Chem. 260(28), 15168-15171 (1985), that methylene blue oxidizes glutathione directly when it is reduced by NADPH, rather than via the H 2 O 2 .
• U.S. Ser. No. 264,088 entitled "Phototherapy Using Methylene Blue” filed Oct. 28, 1988 by Robert A. Floyd disclosed a method for using thiazine dyes, especially methylene blue, in combination with light to hydroxylate guanosine or deoxyguanosine at the C8 of the purine ring.
• the number of guanosines in a nucleic acid strand converted to 8-OH-deoxyguanosine (8-OH-dG) or 8-OH-guanosine (8-OH-G) can be controlled through manipulation of the concentration of methylene blue, light intensity and length of exposure, pH, and buffer strength. Very little, if any, other derivatives are formed.
• the method can be used for the selective mutation or modification of either a DNA or a RNA sequence, or the protein expressed therefrom.
• 8-OH-dG and 8-OH-G do not base pair well and are especially susceptible to misreading.
• 8-hydroxydeoxyguanosine is made from guanosine by the action of reagents which generate oxygen radicals, such as ascorbic acid and other reducing agents, metals, polyphenols, and asbestos, and by x-irradiation. Intracellular DNA appears to undergo repair by enzymes following formation of 8-hydroxy-deoxyguanosine. This may be a naturally occurring response which has evolved to combat the effects of the many mutagens, tumor promoters, and carcinogens which cause the formation of 8-hydroxydeoxyguanosine.
• oxygen radicals such as ascorbic acid and other reducing agents, metals, polyphenols, and asbestos
• the selective administration of thiazine dyes can be used in the treatment of viral infections and in cancer.
• Selective delivery can be achieved using systems such as liposomes for delivery to macrophages and other phagocytic cells or biodegradable controlled release implants.
• Viruses, bacteria, and cells undergoing rapid DNA synthesis are all inactivated by methylene blue when irradiated with light. Treatment can be extracorporeal or by light irradiation of specific tissues using other methods.
• Methylene blue absorbs in the red wavelengths, i.e., approximately 670 nm, which penetrates tissue much better than other lower wavelengths.
• AIDS Acquired Immunodeficiency Syndrome
• retrovirus variously termed human T-lymphotropic virus type III (HTLV-III), lymphadenopathy-associated virus (LAV), AIDS associated retrovirus (ARV), or human immunodeficiency virus (HIV-1).
• HTLV-III human T-lymphotropic virus type III
• LAV lymphadenopathy-associated virus
• ARV AIDS associated retrovirus
• HIV-1 human immunodeficiency virus
• a patient is generally diagnosed as having AIDS when a previously healthy adult with an intact immune system acquires impaired T-cell immunity.
• the impaired immunity usually appears over a period of eighteen months to three years.
• the patient becomes susceptible to opportunistic infections, various types of cancer such as Kaposi's sarcoma, and other disorders associated with reduced functioning of the immune system.
• AZT 3'-azido-3'-deoxythymidine
• thiazine dyes examples include methylene blue, azure A, azure C, toluidine blue O, and thionine.
• the preferred dye at this time is methylene blue.
• Methylene blue is FDA approved for topical, i.v., and oral administration, and has minimal side effects. Selective delivery can be achieved using systems such as liposomes for delivery to macrophages and other phagocytic cells or using biodegradable controlled release implants. Since methylene blue absorbs in the red wavelengths, i.e., approximately 670 nm, which penetrates tissue much better than other lower wavelengths, light penetrating the skin to the capillaries at the surface can be used to enhance the activity of the dye.
• FIGS. 1A and 1B are graphs of the effect of methylene blue in the light and in the dark on the replication of HIV-1 in human PBM cells, % inhibition versus log concentration of methylene blue, ⁇ M,
• FIG. 1A is a bar graph;
• FIG. 1B is a line graph.
• the production of 8-OH-G results in mutations in nucleic acid since it does not base-pair as well as the unaltered guanosine and because the bases adjacent to the 8-OH-G can be misread during replication, transcription, and translation.
• Viruses replicate within the host's cells, using enzymes encoded by their own genetic material and the host cell's "machinery". Replication usually occurs at a high rate, with much of the transcription and translation being involved in production of new virus. It has now been discovered that the dyes interfere with transcription and translation in the dark or in low light levels, as well as in light. Further, it has been discovered that some types of viruses are particularly sensitive to the thiazine dyes.
• viruses examples include human immunodeficiency virus (HIV), and, to a lesser degree, Herpes simplex virus (HSV).
• HSV Herpes simplex virus
• the thiazine dyes can be used to treat infections by these viruses in vivo by interfering with the transcription and translation involved in replication of the virus in the host cells, at least in part by the mechanism whereby the dye hydroxylates the guanosine or deoxyguanosine to yield 8-OH-guanosine (8-OH-G) or 8-OH-deoxyguanosine (8-OH-dG), respectively.
• 8-OH-G 8-OH-deoxyguanosine
• 8-OH-dG 8-OH-deoxyguanosine
• the activity of the dye can be enhanced further by irradiation with light or by derivatization with compounds such as antisense mRNA.
• the thiazine dye can also be provided in combination with other known antibiotics, anti-inflammatories, antifungals, and antivirals.
• Thiazine dyes have been used in a variety of applications, as discussed in the Background of the Invention.
• the method described herein is based on the selective and controlled use of the compound, not the complete destruction of all genetic material, which would be completely inapplicable to any method wherein the goal is to preserve and minimize toxic effects to the host cells.
• the method described herein does not require administration of exogenous light, although the results may be enhanced by exposure to light in addition to that normally transmitted through the skin.
• thiazine dyes examples include methylene blue, azure A, azure B, toluidine blue O, and thionine. Methylene blue is the preferred dye at this time. These dyes are all commercially available from a number of different sources.
• the dyes can be applied topically or systemically for in vivo applications. Both methods of administration are approved by the Federal Drug and Food Administration for methylene blue, although methylene blue is not FDA approved at this time for any topical or in vivo application for the treatment of viral infections.
• Methylene blue is a thiazine dye occurring as dark blue-green crystals which is soluble in water and sparingly soluble in alcohol, forming deep blue solutions.
• Methylene blue injectable has a pH of 3-4.5. The pK a is between 0 and -1.
• the 24 h LD 50 for intravenous methylene blue administered as a 3% solution was 42.3 mg/kg with 95% confidence interval limits of 37.3 to 47.9 mg/kg, demonstrating that methylene blue can be safely administered at a dosage of up to at least 15 mg/kg.
• methylene blue crosses the blood-milk barrier easily.
• the method described herein for the inhibition of HIV infections in vivo requires dosages in the range producing a blood concentration of approximately 20 to 200 micromolar, or 7.5 to 75 mg/l.
• the usual blood volume for babies is approximately 2.5 l, for adult humans it is approximately 10 l.
• this is approximately equivalent to 5.76 mg/kg over an 18 hour period.
• the thiazine dyes can also be delivered using techniques known to those skilled in the art of drug delivery to target specific cell types or to enhance the activity of the dye. For example, a procedure utilizing injection of photoactive drugs for cancer treatment is described by Edelson, et al., in New England J. Med. 316, 297-303 (1987). Thiazine dyes can be specifically delivered to macrophages, a site of high HIV concentration in AIDS patients, using techniques such as liposome delivery Liposomes are generally described by Gregoriadis, Drug Carriers in Biology and Medicine Ch. 14, 287-341 (Academic Press, N.Y., 1979). Methods for making light sensitive liposomes are described by Pidgeon, et al., in Photochem. Photobiol.
• Liposome compositions are commercially available from companies such as the Liposome Company, Inc., Princeton, N.J. Release of compounds from liposomes ingested by macrophages is described by Storm, et al., in Biochim. Biophys. Acta 965, 136-145 (1988).
• the dye can be continuously delivered to a patient over an extended period of time using a controlled release polymeric implant.
• Polymeric implants are generally manufactured from polymers which degrade in vivo over a known period of time. Examples of useful polymers include polyanhydrides, polylactic acid, polyorthoester, and ethylene vinyl acetate. These devices are also commercially available. Alza Corporation, Palo Alta, Calif., and Nova Pharmaceuticals, Baltimore, Md., both manufacture and distribute biodegradable controlled release polymeric devices.
• Methylene blue plus light causes formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG).
• 8-OHdG 8-hydroxy-2'-deoxyguanosine
• Methylene blue was added to calf thymus DNA solubilized in sodium phosphate buffer (0.0935 mg DNA/ml, 0.014M Na Phosphate, pH 6.8) to produce a final concentration of 0.02 mM.
• the methylene blue containing DNA solution was then exposed to white light (100 watt incandescent light 11 cm from a beaker containing the sample being treated) in an ice bath.
• the treated DNA was precipitated with ethanol (400 ⁇ l 5M NaCl in 5 ml ethanol), collected by centrifugation at 2000 rpm for one hr at 4° C., and the pellet redissolved in 250 ⁇ l Bis-Tris EDTA buffer containing 10 mM MgCl 2 .
• the redissolved DNA was next placed in a boiling water bath for three min then cooled rapidly, digested with DNAse I (10 ⁇ g) and endonuclease (0.6 units) overnight at 37° C.
• the DNA was further digested to the free nucleotide level with phosphodiesterase (PDE) (Calbiochem snake venom, 0.04 units) and alkaline phosphatase from calf intestine (Calbiochem, 1 unit) overnight at pH 8.0 and 37° C.
• PDE phosphodiesterase
• the pH of the solution was lowered to 4.0 with acetic acid and the volume was adjusted to 250 ⁇ l.
• the sample was filtered and run on HPLC to measure the concentration of 8-hydroxyguanosine. Methylene blue plus light clearly causes hydroxylation of guanine as shown in Table 1.
• nucleosides of adenine, cytosine, thymine, and guanine were also subjected to methylene blue (20 ⁇ l into 2.0 mls final total volume) plus light treatment (100 watt incandescent light 11 cm over solution for 15 min). Only deoxyguanosine reacted to form the hydroxylated derivative, based on HPLC analysis.
• RNA virus R17 Exposure of the RNA virus R17 to 0.02 ⁇ M methylene blue plus light for 15 min (100 watt incandescent bulb at 11 cm) causes inactivation of R17, as assessed by its ability to form plaques on a bacterial lawn. The infectivity of the virus is inactivated 50% by a 45 seconds exposure to light in the presence of 0.05 ⁇ M methylene blue.
• viable R17 phage were titered by making serial 10x dilutions in the microtiter plate, adding 0.1 ml of selected dilution to 0.2 ml of log phase E. coli strain XL-1-Blue cells (approximately 10 7 cells/ml), obtained from Stratagene, La Joya, Calif., and plating out 0.1 ml.
• the methylene blue did not inhibit growth of the bacterial lawn at the concentrations used in the R17 inactivation and titering.
• R17 virus samples were also tested to determine the time of exposure of light required to inactivate the virus.
• the procedure was similar to that used for Example 2 as outlined above except that the concentration of MB was held constant at 0.05 uM while the time of exposure was varied.
• the length of exposure was varied as follows:
• AZT was synthesized and purified by a modification of the method of Lin and Prusoff (Lin, T. -S, and W. H. Prusoff, J. Med. Chem. 21, 109-112 (1978).
• Acyclovir (ACV) was obtained from the Burroughs-Wellcome Co.
• PBMC peripheral blood mononuclear cells
• PBMC Human peripheral blood mononuclear cells
• PBS phosphate-buffered saline
• PHA phytohemagglutinin
• Retrovirus HIV-1 (strain LAV-1) was obtained from Dr. P. Feorino (Centers for Disease Control, Atlanta, Ga.). The virus was propagated in human PBMC using. RPMI 1640 medium, as described previously by McDougal, et al., ("Immunoassay for the detection and quantitation of infectious human retrovirus, lymphadenopathy-associated virus (LAV)," J. Immun. Meth.
• the drugs at twice their final concentrations in 5 ml of RPMI 1640 medium (supplemented as described above), were added to the cultures. Uninfected and untreated PBMC at equivalent cell densities were grown in parallel as controls. The cultures were maintained in a humidified 5% CO 2 -95% air incubator at 37° C. for six days after infection at which point all cultures were sampled for supernatant RT activity. Previous studies had indicated that maximum RT levels were obtained at that time.
• RT Activity Assay A six milliliter aliquot of supernatant from each culture was clarified of cells at 300 ⁇ g for 10 minutes. Virus particles were pelleted from 5 ml samples at 40,000 rpm for 30 minutes using a Beckman 70.1 Ti rotor and suspended in 200 ⁇ l of virus disrupting buffer (50 mM Tris-HCl, pH 7.8, 800 mM NaCl, 20% glycerol, 0.5 mM phenylmethyl sulfonyl fluoride, and 0.5% Triton X-100).
• virus disrupting buffer 50 mM Tris-HCl, pH 7.8, 800 mM NaCl, 20% glycerol, 0.5 mM phenylmethyl sulfonyl fluoride, and 0.5% Triton X-100.
• RT assay was performed in 96-well microtiter plates, as described by Spira, et al. (1987), "Micromethod for Assaying the Reverse Transcriptase of LAV-HTLV-III/Lymphadenopathy-Associated Virus," J. Clin. Microbiol. 25, 97-99.
• the reaction mixture which contained 50 mM Tris-HCl, pH 7.8, 9 mM MgCl 2 , 5 mM dithiothreitol, 4.7 ⁇ g/ml (rA) n .(dT) 12-18 , 140 ⁇ M dATP, and 0.22 ⁇ M 3 H!TTP (specific activity 78.0 Ci/mmol, equivalent to 17,300 cpm/pmol; NEN Research Products, Boston, Mass.), was added to each well. The sample (20 ⁇ l) was added to the reaction mixture which was then incubated at 37° C. for 2 hours. The reaction was terminated by the addition of 100 ⁇ l 10% trichloroacetic acid (TCA) containing 0.45 mM sodium pyrophosphate.
• TCA trichloroacetic acid
• the acid-insoluble nucleic acids which precipitated were collected on glass filters using a Skatron semi-automatic harvester.
• the filters were washed with 5% TCA and 70% ethanol, dried, and placed in scintillation vials.
• Four ml of scintillation fluid (Econofluor, NEN Research Products, Boston, Mass.) were added and the amount of radioactivity in each sample was determined using a Packard Tri-Carb liquid scintillation analyzer (model 2,000CA). The results were expressed in dpm/ml of original clarified supernatant.
• the procedures for the anti-HIV-1 assays in PBMC described above have been published recently (see Schinazi, et al., Antimicrob. Agents Chemother. 32, 1784-1789, December 1988).
• HSV Preparation Confluent HEp-2 cells, in a roller bottle (Falcon, 850 cm 2 ), were infected at an input multiplicity of 0.01 PFU per cell (to minimize production of defective viruses). During the two-hour absorption period, the cells were exposed to the virus inoculum diluted in absorption buffer (10 ml PBS containing 1% newborn calf serum and 0.1% glucose). The virus inoculum was then removed and replaced with Hanks' Minimum Essential Medium (MEM) containing 2% inactivated newborn calf serum, penicillin (100 U/ml) and streptomycin (100 ⁇ g/ml) (P and S), sodium bicarbonate (2 g/L) and HEPES (25 mM) (maintenance medium).
• MEM Hanks' Minimum Essential Medium
• the infected cells were incubated at 37° C. for 3 to 4 days, until the cells could be easily shaken from the plastic surface of the culture bottles.
• the cells were then collected by centrifugation, suspended in a small volume of spent culture fluid (5 ml per 2 ⁇ 10 8 cells) and sonicated 3 times for one min on ice.
• the disrupted cells were centrifuged (2,000 g for 15 min at 4° C. and the supernatant was diluted with an equal volume of sterile skimmed milk (as stabilizer) and aliquots were frozen at -70° C.
• HSV Plaque Reduction Assay Near confluent Vero (African Green Monkey) cells in 6-well plates were infected with 100 ⁇ l virus diluted in absorption buffer to give 100-200 plaques per well. The plates were then incubated at 37° C. for 1 hour with intermittent rocking every 15 minutes. The inoculum was aspirated and the compounds at different concentrations (dissolved in maintenance medium) were added to replicate wells. For these assays, 0.1% pooled human gamma globulin was included in the media.
• the plates were placed in 5% CO 2 -95% air incubator and the plaques were allowed to develop for 48 h prior to fixation (buffered 10% Formalin acetate), staining (0.5% crystal violet in 20% EtOH/H 2 O) and enumeration.
• the degree of inhibition (per cent plaques of control) was calculated by counting the mean plague counts for the different drug dilutions.
• the antiviral potency of the drugs was determined by estimating the ED 50 , the drug concentration necessary to reduce the number of plaques by 50% relative to the virus control cultures.
• HSV-1 For routine antiviral drug screening, we used the F strain of HSV-1 and the G strain of HSV-2 (Ejercito, et al., "Characterization of Herpes Simplex Virus Strains Differing in Their Effect on Social Behavior of Infected Cells," J. Gen. Virol. 2, 357-364 (1968)).
• Vero cells for HSV plaguing, Vero cells (rather than a human cell line such as fibroblasts) were used since these cells do not induce interferon.
• Acyclovir (ACV) was used as a positive control for the studies on HSV.
• Vero cells in growth medium were added to 25 cm 2 flasks (Falcon) in duplicate at a concentration equivalent to one-tenth of cell confluency for each compound under test. After incubation at 37° C. in 5% CO 2 -95% air for 24 hr, the test compound (2x final concentration), dissolved in 2.5 ml of the growth medium was added, and two flasks were harvested immediately by decanting the medium, washing once with 3 ml of PBS, and then incubating at 37° C. for 5 min with 3 ml of trypsin/EDTA (0.125%/0.02%).
• the cells dislodged from the flask were generally in clumps and were dispersed by repeated forceful pipetting of the suspension against the surface of the flask.
• 0.2 ml of trypan blue solution was added, and the number of cells were counted using a hemacytomer.
• Each day for the next 3 days two of the remaining flasks were harvested in the manner just described for determination of cell number. Only data on day three are presented. This method has previously been described by Schinazi, et al., "Effect of Combination of Acyclovir, and Vidarabine or its 5'-monophosphate on Herpes Simplex Viruses in Cell Culture and in Mice," Antimicrob. Acents Chemother. 22, 499-507 (1982)).
• PBM and CEM Cells Proliferation Assay The drugs were evaluated for their potential toxic effects on uninfected PHA-stimulated human PBM cells and also in CEM cells. The cells were cultured with and without drug for 6 days at which time aliquots were counted for cell viability as described above.
• Enzyme Assays The preparation of the enzyme and the assay conditions used, were recently described by Schinazi, et al., Antimicrob. Agents Chemother. 33, 115-117 (1989). HIV-1 RT and cellular DNA polymerase alpha were isolated from infected and uninfected PHA-stimulated human PBM cells according to traditional methods (Eriksson, B., et al., Antimicrob. Agents Chemother. 31, 600-604, (1987); Furman, et al., Proc. Natl. Acad. Sci. USA 83, 8333-8337, (1986); Abrell, et al., J. Virol. 12, 431-439, (1973).
• Peripheral blood mononuclear (PBM) cells were infected with HIV and treated with methylene blue as described above.
• the EC 50 levels were calculated from enzyme immuno-assay (EIA) measurements of viral coat protein P24.
• EIA enzyme immuno-assay
• the P24-specific EIA kit was purchased from Abbott Labs.
• the effectiveness of methylene blue was compared with AZT, the results are shown in Table 5.

Abstract

Description

              TABLE 1                                                     
______________________________________                                    
Quantitation of 8-hydroxyguanosine in DNA                                 
exposed to methylene blue and light.                                      
Experimental    Ratio of 8-OHdG                                           
Condition       to 10.sup.5 dG                                            
______________________________________                                    
DNA + Light     6.8                                                       
DNA + MB        24.2                                                      
DNA + Light + MB                                                          
                484.2                                                     
______________________________________                                    
 Data represent the average of 5 replicate samples                        

              TABLE 2                                                     
______________________________________                                    
Effect of methylene blue and light on the                                 
infectivity of RNA Virus R17.                                             
MB conc.                                                                  
(μM)        Light 5'                                                   
                       pfu/ml                                             
______________________________________                                    
0              no      7.20 × 10.sup.11                             
0.02           yes     5.04 × 10.sup.10                             
0.04           yes     1.11 × 10.sup.10                             
0.2            yes     3.25 × 10.sup.5                              
2.0            no      4.80 × 10.sup.10                             
2.0            yes     3.00 × 10.sup.3                              
20.0           no      3.00 × 10.sup.9                              
20.0           yes     4.20 × 10.sup.4                              
______________________________________                                    

              TABLE 3                                                     
______________________________________                                    
Effect of time of exposure to light on                                    
infectivity of R17 virus treated with MB.                                 
Time (Min.)   PFU                                                         
______________________________________                                    
none          2.76 × 10.sup.9 /ml                                   
0.1'          3.3 × 10.sup.9 /ml                                    
0.5'          5.0 × 10.sup.9 /ml                                    
1.0'          1.0 × 10.sup.9 /ml                                    
2.5'          9.1 × 10.sup.7 /ml                                    
5.0'          6.93 × 10.sup.7 /ml                                   
10.0'         4.35 × 10.sup.6 /ml                                   
______________________________________                                    

______________________________________                                    
Assays. Reaction mixtures (100 μl) contained:                          
              RT           DNA pol. alpha                                 
______________________________________                                    
Tris-HCl, pH 8.0                                                          
                100    mM      100   mM                                   
KCl             50     mM      --                                         
MgCl.sub.2      2      mM      6     mM                                   
DTT             5      mM      5     mM                                   
BSA             400    μg/ml                                           
                               400   μg/ml                             
 .sup.3 H!dTTP, 1      μM   1     μM                                
(Sp. act. 82.3 Ci/mmol)                                                   
dATP, dCTP, dGTP                                                          
                --         100     μM                                  
Poly(rA)-oligo(dT).sub.12-18                                              
                3      μg/ml                                           
                               --                                         
Activated DNA   --         200     μg/ml                               
______________________________________                                    

              TABLE 5                                                     
______________________________________                                    
Effect of MB against HIV (LAV-I)                                          
in human PBMC Determination of P24 Levels by EIA                          
(Abbott Ag Kit)                                                           
           Conc.             % Inhibition                                 
                                     EC.sub.50                            
Treatment  μM    ng P24/ml                                             
                             (corrected)                                  
                                     μM                                
______________________________________                                    
Uninfected control  -0.1                                                  
Infected control    336.0                                                 
                    370.5                                                 
                    370.5                                                 
           mean     359.0    0.0                                          
AZT        0.0001   360.7    -0.5                                         
           0.001    266.2    25.8                                         
           0.01     36.1     89.9    0.0064                               
                                           μM                          
           0.1      5.2      98.5                                         
M.B. Light 0.001    370.5    -3.2                                         
           0.01     370.5    -3.2                                         
           .1       302.2    15.8                                         
           1        5.1      98.6    0.024                                
           10       2.2      99.4                                         
M.B. Dark  0.001    356.0    0.8                                          
           0.01     270.2    24.7                                         
           .1       287.2    20.0                                         
           1        84.1     76.6    0.33                                 
           10       5.4      98.5                                         
______________________________________                                    

              TABLE 6                                                     
______________________________________                                    
Effect of MB on HIV-1 reverse transcriptase                               
(RT) and calf thymus DNA polymerase in the                                
presence and absence of light.                                            
               IC.sub.50 (μM)                                          
Enzyme           Light  Dark                                              
______________________________________                                    
HIV-1 RT         9.2    10.0                                              
CT α pol.  125    105.1                                             
______________________________________                                    

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